Action of the Protease from Streptomyces cellulosae on L-Leu-Gly

Transcription

1 /. Biochem. 99, (1986) Action of the Protease from Streptomyces cellulosae on L-Leu-Gly Tetsuo MURO, Yoshio TOMINAGA, and Shigetaka OKADA Osaka Municipal Technical Research Institute, Joto-ku, Osaka, Osaka 536 Received for publication, December 16, 1985 The protease from Streptomyces cellulosae preferentially catalyzed the formation of (L-Leu-Gly), (Pj) and (L-Leu-Gly) 3 (P 2 ) in highly concentrated solutions of L-Leu- Gly, although it weakly hydrolyzed the substrate at the same time. The formation of P 1; P 2, L-Leu, and Gly was studied at various ph values, temperatures, and substrate concentrations. The initial velocities (v lt v 2, and v H ) of formation of P 1; P 2, and L-Leu (or Gly) and the sum (v lt ) of v x and v 2 were determined. The effects of ph and temperature on v x, v 2, v H, and v lt were examined at a fixed substrate concentration. The optimum ph and optimum temperature for each of the processes forming P lt P», L-Leu, and Gly were 8.0 and 65 C, respectively. In the study on the effect of substrate concentration, the plots of the initial velocities versus substrate concentrations were sigmoidal at lower substrate concentrations. The dependence of v lt on the substrate concentration could be explained by a mechanism involving a single active center forming the peptide bonds and two substrate-binding sites located on the left sites (S x and S 2 ) and the right sites (S/ and S 2 ') of the active center of this enzyme. Since Bergmann and Fraenkel-Conrat first demonstrated that peptide or amide bond formation could be catalyzed by the reverse reaction of a protease (/), numerous reports have accumulated on the reverse reactions of proteases (2-8). However, there are only a few reports relating to the kinetic study (9) and mechanism (10-12) of enzymatic peptide syntheses. In a preceding paper (13), we showed that the protease from Streptomyces cellulosae catalyzed Abbreviations: P,, (L-Leu-Gly).; P 2, (L-Leu-Gly) 3 ; L, L-Leu; G, Gly; v,, initial velocity of formation of (L-Leu- Gly^; v 2, initial velocity of formation of (L-Leu-Gly) 3 ; v, T, the sum of V! and v 2 ; v n, initial velocity of formation of L-Leu (or Gly). the condensation reaction to produce (L-Leu-Gly) 2 (Pi) and (L-Leu-Gly) 3 (P 2 ) at high concentrations of L-Leu-Gly. The amount of P 2 was always much larger than that of P v L-Leu and Gly were also formed in the course of the condensation reaction and were more evident at lower substrate concentrations. The purpose of the present study was to investigate quantitatively the effects of ph, temperature, and substrate concentration on the formation rates (v lt v 2, v lt, and v H ) of P 1; P 2, the sum of P 2 and P 2, and L-Leu (or Gly) formation by quantitating the products in order to obtain information about the mechanism of the condensation reaction by this enzyme. Vol. 99, No. 6,

2 1626 T. MURO, Y. TOMINAGA, and S. OKADA EXPERIMENTAL PROCEDURES Materials L-Leu-Gly was purchased from Sigma Chemical Co. The protease from Streptomyces cellulosae was purified to an electrophoretically homogeneous state (14). The molar concentration of the enzyme was determined at 280 nm assuming an E value of 3.9 for a 1 % solution in a 1-cm cell, and a molecular weight of 22,000 (14). All other chemicals used were of reagent grade. Methods Turbidity-forming activity of this protease was evaluated by turbidity measurement in a 16% soybean protein hydrolysate (14). The effects of ph and temperature on the initial velocities (v u v 2, and v H ) of formation of (L-Leu-Gly) 2 (Pj), (L-Leu-Gly) 3 (P 2 ), and L-Leu (or Gly) and the 10 sum (v lt ) of V]_ and v 2 were examined at a fixed ph substrate concentration (130 HIM), Fig. 1. The ph dependences of the initial velocities of In kinetic experiments, 0.25 ml of the enzyme formation of (L-Leu-Gly),!, (L-Leu-Gly) 3, and L-Leu solution was added to 0.75 ml of 2.6-1,364 HIM (or Gly) from L-Leu-Gly. A, V,. (initial velocity of L-Leu-Gly in 0.01 M phosphate buffer, ph 8.0 (the (L-Leu-Gly) 2 formation);, v 2 (initial velocity of (Loptimum ph). After an appropriate time of incubation Leu-Gly) 3 formation); O, v 1T (the sum of V! and v 2 ); at a fixed temperature of 25 C, 0.2 ml of the reaction mixture was withdrawn and immediately poured into 0.2 ml of a mixture (1:1, v/v) D, v H (initial velocity of L-Leu (or Gly) formation). In condensation and hydrolytic reactions, the relative velocities of 100% correspond to v 1T and v H at ph 8.0, respectively. Enzyme concentration: 3 x 10" of 1.5 M HCl and 0.4 M phosphate buffer (ph 2.1) M. Substrate concentration: 1.3 x 10" 1 M. to stop the reaction. This solution was subjected to reversed-phase high-performance liquid chromatography (HPLQ for determination of the amounts of T x and P 2, and to amino acid analysis for L-Leu and Gly. The HPLC was carried out with a 5-/<m LiChrosorb respectively. Temperature Dependence of kn The temperature dependence of the rate constant (& lt ), which is equal to K lt /[E] 0, was studied at ph8.0 between RP-18 column (0.5 x 25 cm) at 1 ml/min and 40 C, 20 C and 40 C. V lt is the sum of V,. and V 2, using 0.1M phosphate buffer (ph2.1) containing which are the maximum velocities of formation of 20 % or 30 % acetonitrile as the mobile phase. The Pi and P 2, respectively, and [E] o is the total concentration elution pattern was monitored at 220 nm. of the enzyme. The Arrhenius plots for fc lt are shown in Fig. 3. This plot was linear RESULTS AND DISCUSSION in the range of temperatures measured. From the slope, the activation energy was calculated to Effects of ph and Temperature on v lt v 2, VH, and v lt The effects of ph and temperature on the initial velocities (vj, v 2, and v H ) of formation of (L-Leu-Gly):. (PJ, (L-Leu-Gly) 3 (P 2 ) and L-Leu (or Gly) and the sum (v lt ) of v x and v 2, determined at a substrate concentration of 130 mm, are shown in Figs. 1 and 2, respectively. The optimum ph and optimum temperature for all the processes forming P l5 P 2, L-Leu, and Gly were 8.0 and 65 C, be 6.1 kcal/mol. The free energy change (AG*), the enthalpy change (AH*), and the entropy change (AS*) were calculated to be 18.5 kcal/mol, 5.5 kcal/ mol, and 43.6 e.u., respectively. Dependences of v lt v 2, v H, and v lt on Substrate Concentration The time courses of formation of PL P 2, L-Leu, and Gly, and that of the sum of Pi and P 2, are shown in Fig. 4, which indicates that the values of v 2 were about five times those of /. Biochem.

3 ACTION OF S. cellulosae PROTEASE ON L-Leu-Gly Temperature ( C) Fig. 2. The temperature dependences of the initial velocities of formation of (L-Leu-Gly) 2, (L-Leu-Gly) 3, and L-Leu (or Gly) from L-Leu-Gly. A, v x (initial velocity of (L-Leu-Gly) 2 formation);, v 2 (initial velocity of (L-Leu-Gly) 3 formation); O, v 1T (the sum of Vj and v 2 );, v a (initial velocity of L-Leu (or Gly) formation). In condensation and hydrolytic reactions, the relative velocities of 100% correspond to v 1T and v H at 65 C C, respectively. Enzyme concentration: 3.0 x 10~ 5 M. Substrate concentration: 1.3 x 10~' M. EM /T X 10 Fig. 3. The temperature dependence of k ir at ph 8.0. T is the absolute temperature. Enzyme concentration: 1.4xlO- 5 M. Reaction Time (h) Fig. 4. An example of the time courses of formation of (L-Leu-GIy) 2, (L-Leu-Gly) 3, L-Leu, and Gly. A, (L-Leu-Gly) 2 ;, (L-Leu-Gly) 3 ; O, sum of (L-Leu-Gly) 2 and (L-Leu-Gly) 3 ;, L-Leu; D, Gly. Enzyme concentration: 1.4x 10~ 5 M. Substrate concentration: 1.3 x 10-1 M n 3>. 0 V 0.2 i a.. i. 0 "' a S ( M ) Fig. 5. The effect of substrate concentration on the initial velocities of formation of (L-Leu-Gly) 2, (L-Leu- Gly) 3, and L-Leu (or Gly). The solid line is a theoretical curve drawn according to Eq. 1 with A" a =0.016M, A" b =0.65M, and K,T=2.4X 10" M-S" 1 (see text). A, v, (initial velocity of (L-Leu-Gly) 2 formation);, v 2 (initial velocity of (L-Leu-Gly) 3 formation); O, v, T (the sum of v, and v,.); O, v n (initial velocity of L-Leu (or Gly) formation). Enzyme concentration: 1.4x 10~ 5 M. Vol. 99, No. 6, 1986

4 1628 T. MURO, Y. TOMINAGA, and S. OKADA v x and the values of v H were much smaller than those of v x and v 2 at this substrate concentration. The effects of substrate concentrations on v 1; v 2, v lt, and v H are illustrated in Fig. 5, which clearly shows that the curves for v x> v 2, and v lt are sigmoidal at lower substrate concentrations (2-130 ITIM), whereas v H showed a simple saturation curve over the lower range of substrate concentrations (2-380 mm) and decreased at higher substrate concentrations (above 380 mm). From these results, it can be considered that the effect of v H on v lt v 2, and v lt is negligible, because the values of v H were much smaller than those of the other initial velocities (v lt v 2, and v lt ). Furthermore, v lt v 2, and v lt were little affected by the reverse reaction of P x formation at least at the initial stage of the condensation reaction. Consequently, it can be assumed that the hydrolytic reaction does not affect the initial velocities of formation of P t and P 2. The following mechanism (Scheme 1), involving two substrate-binding sites (sites L and R) in addition to one catalytic site, was able to account for the experimental data: L-G-L-GA site L L-G site R L-G-L-G site L site R L-G-L-G (P x ) Scheme 1 L-G-L-G-L-G (P 2 ) L-G-L-G-L-G (P 2 ) where L-G, L-G-L-G, and L-G-L-G-L-G are L- Leu-Gly, P 1; and P 2, respectively, and S and E are the substrate and enzyme, respectively. Analysis of the Curve ofv lt From the finding that the amount of P 2 was always much larger than that of P x at a definite reaction time (Fig. 4 and Fig. 5), it can be speculated that the rate of the reaction producing P 2 from Pj and L-Leu-Gly is much faster than that of P x formation. It can be also speculated that a large portion of P x produced is immediately utilized for P 2 formation by "sliding" on the enzyme active site or by dissociation-association without moving into the bulk solution at the initial stage of the enzyme reaction. Based on the above speculation, the sum (v lt ) of v x and v 2 may reflect the total rate of formation of Pj. Moreover, it can be considered that v^ represents the approximate velocity of the condensation reaction, since no other condensation products, except P x and P 2, were produced, and the effect of v H on v x and v 2 was negligible. A substrate molecule is bound to site L or site R in two modes to form either complex SE or ES. Furthermore, another substrate molecule is bound to site L of ES or site R of SE to form the ternary complex, SES. The SES can break down into P t and enzyme (E) with rate constant, A- lt. It is also assumed that the binding of substrate to site L does not affect the binding at site R, and vice versa. The following Scheme 2 can be written based on these assumptions: Scheme 2 E + P, where K K and K b are the dissociation constants defined by the following equations: *a = [S][E]/[SE]=[S][ES]/[SES] K b = [E][S]/[ES] = [SE][S]/[SES]. A'a stands for the substrate dissociation constant at site L for complex SE or SES, and K b for the substrate dissociation constant at site R for complex ES or SES. Based on these assumptions, rapid-equilibrium treatment leads to the following rate equation: v lt = * lt [SES] = K lt + S) (K b + S) (1 ) where K lt (=&IT[E] 0 ) is the maximum velocity. The rate equation, Eq. 1, includes the term S 2 in its numerator and denominator. Therefore, it does not follow Michaelis-Menten kinetics, but will show a sigmoidal curve in the v^ versus S plots (Fig. 5). Let us next consider some limiting cases of Eq. 1. J. Biochem.

5 ACTION OF 5. cellulosae PROTEASE ON L-Leu-Gly 1629 (i) At high substrate concentrations where either S»/sT a or S>tf b holds. If S>.Ka holds, the term, K a + S, of the denominator in Eq. 1 may be close to S, and then Eq. 1 is reduced to the Michaelis-Menten type of equation as shown below: Accordingly, the plots of S/v lt versus S should be linear, and the intercept on the horizontal axis gives K b which corresponds to the apparent Michaelis constant. The intercept on the ordinate gives -KV^IT- Figure 6A shows that the linearity between S/VXT versus S holds for substrate concentrations above 130 mm in accordance with Eq. 2. The values of K b and K lt obtained from this plot were about 0.65 M and 2.4 x 10~ 6 M-S~\ respectively. (ii) At lower substrate concentrations where either S< Ke. or S K b holds. If S< K b holds, the term, K b + S, of the denominator in Eq. 1 may be close to K b, and then Eq. 1 is reduced to the following equation: (3) Therefore, the plots of S 2 /VIT against S should be linear, and the intercepts on the abscissa and ordinate give -A" a and K a K b /V lt, respectively. Figure 6B shows that the linearity between S 2 / v lt versus S holds for substrate concentrations below 130mM in accordance with Eq. 3. The values of K a and K e.k b IV l T obtained from this plot were M and 4.3 xlo 3 M-S, respec- Fig. 6. S/v 1T versus S plots (A) and S! / V IT versus S plots (B) for the sum of (L-Leu-Gly) 2 and (L-Leu-G!y) 3. Enzyme concentration: 1.4 x 10~ 6 M. tively. Furthermore, the value of K b was calculated to be M, using the value (2.4 xlo" 6 M-S" 1 ) of F lt obtained from the plots in Fig. 6A and that (0.016 M) of K &. This calculated value of K b was close to the value of K b obtained from the plots in Fig. 6A. The solid line in Fig. 5 was drawn according to Eq. 1 by using the values, ii: a = 0.016M, K b = 0.65 M, and K lt = 2.4x 10-" M- s" 1. The experimental data are well represented by this theoretical curve. Based on the above assumptions, the values of Ki and A" b were obtained as M and 0.65 M, respectively. However, there is no reason to draw any distinction between K & and K b in this analytical method, though it could be considered that either the value of A" a or that of K b should be M or 0.65 M. AS mentioned above, the kinetic data obtained from the analysis of the curve of v lt were successfully explained by Scheme 2 with two substrate-binding sites and one active site on the enzyme. Further study is needed to elucidate the mechanism of the consecutive reaction producing P 2 from P x and L-Leu-Gly, since analysis of the curves of v t and v 2 obtained in this experiment does not provide sufficient information. The authors are very grateful to Prof. K. Hiromi, Faculty of Agriculture, Kyoto University, for valuable suggestions and discussions and for his encouragement. REFERENCES 1. Bergmann, M. & Fraenkel-Conrat, H. (1937) J. Biol. Chem. 124, Inouye, K., Watanabe, K., Morihara, K., Tochino, Y., & Kanaya, T. (1979) /. Am. Chem. Soc. 101, Morihara, K., Oka, T., & Tsuzuki, H. (1979) Nature 280, Masaki, T., Fujihashi, T., Nakamura, K., & Soejima, M. (1981) Biochim. Biophys. Ada 660, Homandberg, G.A. & Chaiken, I.M. (1980) /. Biol. Chem. 255, 4903^ Homandberg, G.A., Komoriya, A., & Chaiken, I.M. (1982) Biochemistry 21, Fujimaki, M., Yamashita, M., Arai, S., & Kato, H. (1970) Agric. Biol. Chem. 34, Yamashita, M., Arai, S., Aso, K., & Fujimaki, M. (1972) Agric. Biol. Chem. 36, Gawron, O., Glaid, A.J., III, Boyle, R.E., & Odstrchel, G. (1961) Arch. Biochem. Biophys. 95, Vol. 99, No. 6, 1986

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